1. Field of the Invention
This invention relates to microfluidic devices for investigating crystallization.
2. Description of the Related Art
Although protein crystallography can be a very successful technique for structure determination, membrane proteins continue to present challenges to crystallization. It has been reported that two thirds of purified proteins fail to produce diffraction quality protein crystals. Of the human membrane proteins, representing one third of the genome, only a few have had their structure solved using X-ray diffraction. In many cases, the number of crystallization trials is limited by the availability of human protein, which does not express well in bacteria, hence the drive to minimize sample volume.
The paradigm guiding many crystallization efforts is that the conditions for which an equilibrium crystal phase exists are a small subset among a vastly larger set of parameters such as protein concentration, pH, various salts, polymers, temperature, and surfactants. However, it is not widely appreciated that finding the correct equilibrium conditions, while a necessary condition, is not sufficient to produce crystals because crystallization is a non-equilibrium process. Consequently, crystallization methods that focus on screening large number of conditions were often incomplete. Additionally, it may be helpful to optimize the non-equilibrium kinetics of protein crystallization and exploit the crystals that are produced by these methods in order to obtain high quality diffraction data.
Under many previous methods, protein crystals are produced by trial and error, which necessitates exploring a large number of conditions consuming milligrams of protein. Many methods employed in small non-automated labs require about 1 microliter of solution per trial. Automation with expensive robotics has lowered volumes to the 100 nanoliter (nL) range in some instances. Microfluidic devices can reduce the volume per trial to 1 nL or less in many instances. Such small volumes prove useful to screen conditions. However, when crystals are produced in 1 nL drops, they can be less than 30 microns in diameter, which may be too small for current diffraction methods. Scale-up from microfluidic systems also may involve different physics and can be difficult. Even if large crystals are obtained, then they may be required to be cryoprotected, which can damage crystals. Finally the crystals must be aligned in the x-ray beam in many systems.
U.S. Patent Application Publication No. 2012/0190127 to Fraden describes a Crystal Optimizer that is designed to optimize the crystallization kinetics by systematically varying the kinetic supersaturation profile of the crystallization solution. The technology of U.S. 2012/0190127 can be used to crystallize proteins on the salvage pathway (promising crystals that fail to yield structures), including human membrane G-protein-coupled receptors. Given the paucity of crystallized human membrane proteins and the fact that 50% of marketed drugs target G-protein-coupled receptors, the systems and methods of U.S. 2012/0190127 can impact fields such as structural biology and pharmaceutical development.
In the pharmaceutical field, crystal polymorphism can have dramatic differences in biological activity between two forms of the same drug. For example, a metastable polymorph may have higher solubility that leads to an increase in the absorption rate and bioavailability of a drug administered orally. Synthetic and analytic departments of leading pharmaceutical companies carry out systematic work to detect polymorphism of their drugs and to find intelligent applications of this phenomenon. The systems and methods of U.S. 2012/0190127 can benefit such systematic work in detecting polymorphism of drugs.
The Crystal Optimizer of U.S. 2012/0190127 addressed the problem of crystal creation by determining favorable conditions for crystallization using microfluidics. However, there is still a need for further microfluidic technology improvements that allow for a systematic and reversible kinetic control of crystallization trajectory and diffraction studies of crystallized molecules.